Advances in understanding of plasmid biology through the application of transposon insertion sequencing

Plasmids are extra-chromosomal DNA molecules capable of autonomous replication, stable inheritance in a bacterial population, and horizontal transfer to other bacteria. Plasmids can harbour auxiliary genetic material that contributes to host bacterial fitness, the most prominent example being antimicrobial resistance (AMR) determinants, which remain the greatest threat to modern medicine. Since their discovery in the early 1950s, plasmids have been extensively studied due to their diversity, their capacity to spread between bacterial hosts, and their ability to carry and disseminate multiple AMR genes simultaneously. Recent advances in sequencing technology have transformed plasmid research, with transposon–insertion sequencing (TIS) enabling simultaneous analysis of millions of mutants and providing unprecedented scale, speed and resolution for studying plasmid biology. Here, we briefly outline a recommended methodology for generating plasmid transposon mutant libraries, which can be combined with TIS to investigate plasmid replication, maintenance and conjugation. We further summarise data from nine comprehensive plasmid TIS studies to date on five distinct plasmids, discuss alternative uses for plasmid libraries, challenges and future perspectives.